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Bilirubin and Amyloid-β Peptide Induce Cytochrome c Release Through Mitochondrial Membrane Permeabilization

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Abstract

Background

The pathogenesis of bilirubin encephalopathy and Alzheimer’s disease appears to result from accumulation of unconjugated bilirubin (UCB) and amyloid-β (Aβ) peptide, respectively, which may cause apoptosis. Permeabilization of the mitochondrial membrane, with release of intermembrane proteins, has been strongly implicated in cell death. Inhibition of the mitochondrial permeability is one pathway by which ursodeoxycholate (UDC) and tauroursodeoxycholate (TUDC) protect against apoptosis in hepatic and nonhepatic cells. In this study, we further characterize UCB- and Aβ-induced cytotoxicty in isolated neural cells, and investigate membrane perturbation during incubation of isolated mitochondria with both agents. In addition, we evaluate whether the anti-apoptotic drugs UDC and TUDC prevent any changes from occurring.

Materials and Methods

Primary rat neuron and astrocyte cultures were incubated with UCB or Aβ peptide, either alone or in the presence of UDC. Apoptosis was assessed by DNA fragmentation and nuclear morphological changes. Isolated mitochondria were treated with each toxic, either alone or in combination with UDC, TUDC, or cyclosporine A. Mitochondrial swelling was measured spectrophotometrically and cytochrome c protein levels determined by Western blot.

Results

Incubation of neural cells with both UCB and Aβ induced apoptosis (p < 0.01). Coincubation with UDC reduced apoptosis by >50% (p < 0.05). Both toxins caused membrane permeabilization in isolated mitochondria (p < 0.001); whereas, pretreatment with UDC was protective (p < 0.05). TUDC was even more effective at preventing matrix swelling mediated by Aβ (p < 0.01). UDC and TUDC markedly reduced cytochrome c release associated with mitochondrial permeabilization induced by UCB and Aβ, respectively (p < 0.05). Moreover, cyclosporine A significantly inhibited mitochondrial swelling and cytochrome c efflux mediated by UCB (p < 0.05).

Conclusion

UCB and Aβ peptide activate the apoptotic machinery in neural cells. Toxicity occurs through a mitochondrial-dependent pathway, which in part involves opening of the permeability transition pore. Furthermore, membrane permeabilization is required for cytochrome c release from mitochondria and can be prevented by UDC or TUDC. These data suggest that the mitochondria is a pharmacological target for cytoprotection during unconjugated hyperbilirubinemia and neurodegenerative disorders, and that UDC or TUDC may be potential therapeutic agents.

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Acknowledgements

This work was supported by grant PRAXIS/C/SAU/14311/1998 from Fundaçào para a Ciência e a Tecnologia, Lisbon, Portugal, and EASL Research Fellowship from the European Association for the Study of the Liver to CMP Rodrigues.

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  1. Centro de Patogénese Molecular, Faculdade de Farmácia, University of Lisbon, Av. Forças Armadas, 1600-083, Lisbon, Portugal

    Cecília M. P. Rodrigues Ph.D., Susana Solá, Rui Silva & Dora Brites

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  1. Cecília M. P. Rodrigues Ph.D.
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  2. Susana Solá
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Correspondence to Cecília M. P. Rodrigues Ph.D..

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Rodrigues, C.M.P., Solá, S., Silva, R. et al. Bilirubin and Amyloid-β Peptide Induce Cytochrome c Release Through Mitochondrial Membrane Permeabilization. Mol Med 6, 936–946 (2000). https://doi.org/10.1007/BF03401828

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